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Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02364-2) contains supplementary material, which is available to authorized users.

Polyunsaturated fatty acids are important signaling molecules. A recent study reported hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid, 12-oxo-5Z,8E,10E-heptadecatrienoic acid, and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid as chemotherapy resistance-inducing factors when tumor cells were treated with cisplatin. Marine-based food supplements like fish oil or algae extracts are rich in polyunsaturated fatty acids and can contain large amounts of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid. Thus, it was concluded that oral uptake of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid might induce chemoresistance as shown in a mouse model. Cancer patients tend to consume food supplements containing polyunsaturated fatty acids on a regular basis. The uptake of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid should be controlled, because even low concentrations of 0.5 ng mL-1 showed chemoresistance-inducing effects in animal experiments. For accurate analysis of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid a validated method was developed by using ultrahigh-performance liquid chromatography hyphenated to quadrupole time of flight mass spectrometry via electrospray ionization and sample preparation by solid-phase extraction (SPE) with 3-aminopropyl silica. A combined targeted/untargeted approach was utilized using MS/MS by data-independent acquisition with SWATH and applied to commercial food supplements (refined fish oil, fish oil capsules, algae oil capsules, and flaxseed capsules). Accurate quantification of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid on the MS/MS level with simultaneous untargeted fatty acid screening revealed additional information. The LODs for hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid were 0.036 ng mL-1 and 0.054 ng mL-1, respectively. Since hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid was present in the samples in large amounts and (12S)-hydroxy-5Z,8E,10E-heptadecatrienoic was not expected to be present in high concentrations, two calibration ranges, namely, 0.5–20 ng mL-1 and 5–200 ng mL-1, were validated. An untargeted screening identified 18–39 free fatty acids being present in the lipid extracts of the food supplement samples.

This article provides a condensed introduction to principles of chiral separation, gives a historic overview of the genesis of the most important concepts regarding chiral stationary phase (CSPs), and summarizes the state of the art in a concise manner. Some recent developments in the field of polysaccharide CSPs are outlined. Finally, the article focusses on the new concept of zwitterionic chiral stationary phases and their application profile and peculiarities. Some other trends in column technology, including sub-2 μm and core-shell CSP particles and the emerging field of (UP)SFC, are briefly discussed.

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

Immobilization of enzymes on mesoporous microparticulate carriers has traditionally been accompanied by reduction in enzyme activity. Herein, we document that immobilization of pepsin via amide coupling on gold nanoparticles (GNPs) with a carboxy-terminated hydrophilic PEG7 shell resulted in a heterogeneous nanobiocatalyst with essentially equivalent turnover rates, k cat (90 %), and enhanced catalytic efficiencies, k cat/K M (107 %), compared to homogeneous catalysis with pepsin in free solution for cytochrome C as model substrate. This heterogeneous catalyst showed further at least equivalent bioactivity in a digestion reaction of a protein mixture consisting of cytochrome C, bovine serum albumin, and myoglobin. UHPLC-ESI-QTOF-MS/MS analysis of the digests with subsequent Mascot database search allowed unequivocal identification of all proteins with high score and good sequence coverage. The functionalized nanoparticles were further characterized by Vis spectroscopy in terms of the surface plasmon resonance (SPR) band, by dynamic light scattering (DLS) with regard to hydrodynamic diameters, and in view of their ζ potentials at each step of synthesis and surface modification. These measurements also revealed that the pepsin-functionalized GNPs were sufficiently stable over at least 1 month; thus providing a satisfactory shelf life to the heterogeneous catalyst. Advantageously, the pepsin-GNP bioconjugate can be conveniently removed after reaction by simple centrifugation steps which makes them a useful tool for analysis of therapeutic peptides and proteins, including monoclonal antibodies. The practical utility of the nanobiocatalyst was documented by digestion of a monoclonal antibody which yielded the F(ab')2 fragment with a mass of 97,619.4 Da. Graphical Abstract Pepsin conjugated to pegylated gold nanoparticles exhibit properties of homogeneous catalysis such as enzyme activity like pepsin in solution and ability to pipette the stable colloidal suspension whereas one can take benefit from easy removal by centrifugation and re-use which are characteristics of heterogeneous catalysis.

The presented work deals with the development and comprehensive validation of a quantitative LC-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) method using a triple quadrupole instrument in the MRM mode for the metabolic profiling of amino acids, organic acids, vitamins, some biogenic amines, secondary metabolites of β-lactam antibiotics biosynthesis as well as their intermediates, and degradation products in fermentation broths of β-lactam antibiotics production (in total 57 hydrophilic compounds). A great number of chromatographic systems (22 different stationary phase/mobile phase conditions) were screened for their adequate chromatographic selectivity to cope with isobaric compounds and other critical analyte pairs. Finally, a hydrophilic interaction liquid chromatography (HILIC) method employing a zwitterionic ZIC-HILIC column was selected as best compromise. Particular focus was given on the elucidation of absolute and relative matrix effects via comparison of slopes of calibration functions of spiked matrix and standard solutions. These data as well as precision and accuracy data confirm suitability of the HILIC-ESI-MS/MS assay for metabolic profiling studies in fermentation samples. Detailed comprehensive data sets are presented which should illustrate critical issues, problems, and challenges of multitarget quantitative metabolic profiling and should outline possible strategies to circumvent pitfalls and overcome common problems.

In the present paper, we report on the development of a straightforward reversed-phase liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of the most abundant fatty acids; alpha-tocopherol and cephalosporin P1 in fermentation broths. Using this method, fatty acids could be successfully determined in extracts of fermentation broths from penicillin and cephalosporin production without prior derivatization. Matrix effects were investigated in detail, and various kinds of calibrations (i.e., by use of neat standard solutions as well as by matrix-matched calibration employing standard addition each with and without internal standards) were comparatively assessed. The optimized and validated method was employed for the analysis of extracts of fermentation broths and nutrition media.

A feasibility study on the fast enantioselective two-dimensional HPLC separation of racemic amino acid derivatives is presented. The method involves the on-line coupling of a narrow-bore C18 RP column in the first dimension to a short enantioselective column based on nonporous 1.5 microm particles modified with quinidine carbamate as chiral selector in the second dimension. Conceptually, the system was designed to enable both time-controlled repeated transfer of fractions of the eluate and detector-controlled transfer of selected fractions from column 1 to column 2. To avoid volume overloading of the second chiral column, a narrow-bore reversed phase column was installed in the first dimension. Due to the fast (less than 1.5 minutes) enantiomer separation that occurs in the second dimension, the overall analysis time for the two-dimensional separation of a mixture of nine racemic 3,5-dinitrobenzoyl amino acids was optimized at 16 minutes.

Despite the demonstrated high performance of stereoselective capillary electrochromatography (CEC) [1], the broader acceptance of this appealing separation technique has been strongly impeded by the weakness of the non-dedicated, conventional packed column technology normally employed, which is based on microparticulate materials (3–5 lm) and depends on sintered retaining frits. To alleviate the need for frits and related problems (such as bubble formation, barely reproducible manufacture, fragility) and in particular poor column longevity, researchers’ attempts to improve on the CEC column technology and thereby simplify the practical work have recently focused on monolithic type columns where the continuous porous solid is anchored to the capillary wall. Along this line, three distinct types of enantioselective monolithic CEC columns have evolved [2, 3]. (i) Enantioselective monolithic capillaries that make use of particulate CSPs have been prepared by either embedding the particles (25–30%) in a monolithic bed (such as a sol–gel matrix [4] or a polyacrylamide polymer [5]) or by stabilizing the densely-packed particulate bed via a sol–gel matrix [6]. (ii) Silica monolithbased enantioselective columns are prepared in situ in a capillary using sol–gel technology via steps of gelation and phase separation (macropore formation) and subsequent hydrothermal treatment or aging (mesopore formation), which allows independent control of mesopores and macropores and yields an inorganic material with a bimodal pore distribution and high active surface area [7, 8]. In a subsequent step, the chiral selector is immobilized through either covalent linkage [9] or via adsorptive coating of a polymeric selector [10, 11]. (iii) In contrast to the above approaches, enantioselective organic polymer monoliths may be prepared in a single step from a chiral monomer (a chiral selector with a polymerizable functionality), an ionizable co-monomer for EOF generation, another co-monomer that allows the surface polarity to be adjusted, a crosslinker, and porogens that induce phase separation during polymerization. This approach is very flexible and allows tuning of the polymer in terms of porosity, macropore diameter and surface chemistry, in order to meet the specific requirements of CEC and enantiomer separation. Chirally-functionalized polyacrylamide [12–15], brush-type chiral polymethacrylate [16], and molecularly-imprinted chiral polymer monoliths [17] have been successfully developed for enantioselective CEC by incorporating various different chiral recognition principles.

Herein, we report on the in vitro change of DNA conformation of plasmids bound to a 3-aminopropyl-modified mica surface and monitoring the events by atomic force microscopy (AFM) imaging under near physiological conditions. In our study, we used an intercalating drug, chloroquine, which is known to decrease the twist of the double helix and thus altered the conformation of the whole DNA. During our experiments, a chloroquine solution was added while imaging a few highly condensed plasmid nanoparticles in solution. AFM images recorded after the drug addition clearly show a time-resolved relaxation of these bionanoparticles into a mixture of loose DNA strands.

We recently introduced a mixed-mode reversed-phase/weak anion-exchange type separation material based on silica particles which consisted of a hydrophobic alkyl strand with polar embedded groups (thioether and amide functionalities) and a terminal weak anion-exchange-type quinuclidine moiety. This stationary phase was designed to separate molecules by lipophilicity and charge differences and was mainly devised for peptide separations with hydroorganic reversed-phase type elution conditions. Herein, we demonstrate the extraordinary flexibility of this RP/WAX phase, in particular for peptide separations, by illustrating its applicability in various chromatographic modes. The column packed with this material can, depending on the solute character and employed elution conditions, exploit attractive or repulsive electrostatic interactions, and/or hydrophobic or hydrophilic interactions as retention and selectivity increments. As a consequence, the column can be operated in a reversed-phase mode (neutral compounds), anion-exchange mode (acidic compounds), ion-exclusion chromatography mode (cationic solutes), hydrophilic interaction chromatography mode (polar compounds), and hydrophobic interaction chromatography mode (e.g., hydrophobic peptides). Mixed-modes of these chromatographic retention principles may be materialized as well. This allows an exceptionally flexible adjustment of retention and selectivity by tuning experimental conditions. The distinct separation mechanisms will be outlined by selected examples of peptide separations in the different modes.

A recently reported chiral strong cation exchanger (cSCX) type stationary phase was investigated for the LC separation of a series of Cinchona alkaloids and synthetic derivatives thereof to test its usefulness as alternative methodology for the separation of those important pharmaceuticals. The cSCX column-packing material was qualitatively compared on the one hand against a commercially available non-enantioselective SCX-material, PolySulfoethyl-A, and, on the other hand, against a modern C18 reversed-phase stationary phase which is commonly employed for Cinchona alkaloid analysis. Both SCX columns showed no pronounced peak-tailing phenomena which typically hamper Cinchona alkaloid RP analysis and require specific optimization. Thus, the cSCX-based assay provided new feasibilities for the separation of the Cinchona alkaloids in polar organic mode as opposed to conventional reversed-phase methodologies. In particular, a method for the simultaneous determination of eight Cinchona alkaloids (quinine, quinidine, cinchonine, cinchonidine, and their corresponding dihydro analogs) using the cSCX column in HPLC has been developed and exemplarily applied to impurity profiling of a commercial alkaloid sample. Furthermore, both SCX materials allowed successful separation of C9-epi and 10,11-didehydro derivatives from their respective educts in an application in synthetic Cinchona alkaloid chemistry.